Methods for Producing High Toughness Silk Fibres

ABSTRACT

The present invention provides methods for producing a silk protein spinning dope solution suitable for producing high toughness fibres, the thus produced silk protein spinning dope solution, methods for producing fibres using said silk protein spinning dope solution.

The present invention provides methods for producing a silk proteinspinning dope solution suitable for producing high toughness fibres, thethus produced silk protein spinning dope solution, methods for producingfibres using said silk protein spinning dope solution.

BACKGROUND OF THE INVENTION

Silk is an amazing material produced naturally by various species, suchas the silk moth and silk worm (Lepidoptera), bees, wasps, and ants(Hymenoptera) and spiders (arthropods). Each species' silk has its ownunique set of properties.

For example, silk from the silk moth Bombyx mori is ideally suited forfashion textiles due to its light weight, soft touch, and luxuriousappearance. Although silks from other species, especially spider silk,have even higher toughness and tensile strength, as well as betterchemical resistance—properties that make them of great interest toindustry—they have not been produced commercially to date. Spiders canproduce various kinds of silk—each perfectly adapted to the specificrequirements demanded by nature. Orb-web-spinning spiders produce silkfibres with mechanical properties unmatched in the natural world therebyoutcompeting many synthetic fibres produced by modern technology (Slottaet al. (2012) Chemical engineering process 108, 34-49).

Spider webs can withstand high deformations for example caused by theimpact of prey, due to the interplay between several specialized typesof silk fibres. Dragline (or major ampullate) silk forms the frame andradii of the web and serves as a lifeline for the spider during escape.Flagelliform silk, which is more-elastic, makes up the capture spiral ofthe net. Other silks are responsible for reproductive purposes or asglue substance, among others (Slotta et al. (2012) Chemical engineeringprocess 108, 34-49).

Spiders are able to produce the high-performance polymer material underenvironmentally friendly conditions using aqueous solutions, ambienttemperature, and with low energy consumption. However, the complexmechanisms behind the seemingly simple process of natural threadformation and web construction are not yet understood and thereforecannot be readily replicated.

Many attempts have been made to mimic the spinning process at thelaboratory scale and significant progress has been made, but themechanical properties of the natural dragline fibre are still unmatched.

To produce a commercial fibre, either the natural process of silkspinning must be mimicked, or a completely new spinning process must bedeveloped. To be commercially viable, any process must be cost-efficientand environmentally friendly.

Few data on the mechanical properties of synthetic silk fibres can befound in the literature. Most of the spinning processes create fibresthat are so brittle that their mechanical properties cannot be properlymeasured or the resulting fibres loose performance upon drying orstorage.

However, these studies do provide some useful hints about the keys tospinning silk protein. For instance, a higher-molecular-weight proteinproduces a more-stable fibre, as reported by Xia et al. ((2010) PNAS107: 14059-14063). Here, recombinant proteins originating from thespider Nephila clavipes were produced and spun into a fibre displayingmechanical properties approaching those of native silk. However, suchtoughness was only obtained for proteins of very high molecular weight.For recombinant spider silk proteins with a molecular weight of almost300 kD a fibre exhibiting a toughness of 141 MJ/m³ was obtained. Atlower molecular weight the toughness was far inferior to native silk.These effects may be due to the reported difficulties to retain theprotein at high concentrations, especially in an aqueous system.

The right combination several factors are thought to greatly improve themechanical properties of the spun fibres. However, despite variouspromising approaches, the mechanical properties of natural draglinefibres have not been reproduced before.

The inventors of the present invention surprisingly found a method forproducing an aqueous silk protein spinning dope solution forself-assembling polypeptides, such as spider silk polypeptides. Spinningof this dope results in fibres with a very high toughness, which tendsto increase with molecular weight but is at all molecular weightssuperior to the toughness of silk fibres produced according to themethod disclosed by Xia et. al (supra). Thus, the method of the presentinvention enables for the first time a unique formation of silk proteinsin solution resulting in fibres with a toughness far better thanreported hitherto.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a method forproducing a silk protein spinning dope solution comprising the steps:

-   -   (a) providing an aqueous solution comprising at least one silk        protein and a protein denaturant or mixture of protein        denaturants at a silk protein denaturing concentration, wherein        the total concentration of the silk protein(s) in the solution        is less than 20% w/v;    -   (b) reducing the concentration of the protein denaturant by        8-fold to 14-fold;    -   (c) reducing the concentration of the protein denaturant by 1.5        to 3-fold; and    -   (d) producing the silk protein spinning dope solution by        concentrating the silk protein(s) in the solution at least        1.5-fold in comparison to its(their) concentration in step (a)        to a concentration of at least 10% w/v.

In a second aspect the present invention relates to a method forproducing a fibre comprising the steps:

-   -   (a) providing the silk spinning dope solution producible by the        method of the present invention; and    -   (b) producing a fibre by drawing or extruding or combination        thereof from the silk protein spinning dope solution.

In a third aspect the present invention relates to a spinning dopesolution producible by the method of the present invention.

In a fourth aspect the present invention relates to a fibre producibleby the method of the present invention.

In a fifth aspect the present invention relates to a fibre comprising atleast one silk protein, wherein at least 10% by weight of the materialof the fibre is(are) silk protein(s), wherein the silk proteinmonomer(s) has/have a molecular weight in the range of 20 kDa to 600 kDaand the fibre has a toughness (MJ/m³) that is the product of themolecular weight of the silk protein(s) in kDa and the factor of atleast 1.0 at least up to an molecular weight of the silk protein(s) of300 kDa and is at least 300 MJ/m³ for proteins with an molecular weightof above 300 kDa.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise herein, all technicaland scientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kolbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, GenBank Accession Number sequence submissions etc.),whether supra or infra, is hereby incorporated by reference in itsentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

Residues in two or more polypeptides are said to “correspond” to eachother if the residues occupy an analogous position in the polypeptidestructures. It is well known in the art that analogous positions in twoor more polypeptides can be determined by aligning the polypeptidesequences based on amino acid sequence or structural similarities. Suchalignment tools are well known to the person skilled in the art and canbe, for example, obtained on the World Wide Web, e.g., ClustalW(www.ebi.ac.uk/clustalw) or Align(http://www.ebi.ac.uk/emboss/align/index.html) using standard settings,preferably for Align EMBOSS: needle, Matrix: Blosum62, Gap Open 10.0,Gap Extend 0.5.

Unless otherwise indicated, the terms “polypeptide” and “protein” areused interchangeably herein and mean any peptide-linked chain of aminoacids, regardless of length or post-translational modification.

The term “fibre” refers to a class of materials comprising silk proteinsthat are continuous filaments or are in discrete elongated pieces.

The term “toughness” refers to a property of a fibre that is measure inMJ/m³. It is well know in the art how to measure toughness of a fibre.This can be measured, for example, as described.

As mentioned above, the inventors of the present invention surprisinglyfound that a denatured silk protein solution, if re-natured in acontrolled and unique step-wise fashion and appropriately concentratedleads to a silk protein spinning dope solution in which the proteinsappear to be in a state favoring assembly of the solubilized silkproteins to form a fibre. This is attested to by the fact that thepresent inventors were successful in producing silk fibres ofunprecedented toughness only when using the silk protein spinning dopesolution produced by the method of the present invention. Accordingly,in a first aspect the present invention provides a method for producinga silk protein spinning dope solution comprising the steps:

-   (a) providing an aqueous solution comprising at least one silk    protein and a protein denaturant or mixture of protein denaturants    at a silk protein denaturing concentration, wherein the total    concentration of the silk protein(s) in the solution is less than    20% w/v;-   (b) reducing the concentration of the protein denaturant by 8-fold    to 14-fold;-   (c) reducing the concentration of the protein denaturant by 1.5 to    3-fold; and-   (d) producing the silk protein spinning dope solution by    concentrating the silk protein(s) in the solution at least 1.5-fold    in comparison to its(their) concentration in step (a) to a    concentration of at least 10% w/v.

It is possible to use silk proteins as naturally occurring in the silkfibres of silkworms (e.g. Bombyx mori) or spiders or recombinantlyproduced silk proteins, which may be produced in a suitable host systemcomprising, for example, bacterial cells as, e.g. E. coli, yeast cellsas, e.g. S. pombe, or insect cells as, e.g. Sf9 or Hi5 cells. The silkproteins used in the method of the present invention may be spider silkproteins; insect silk proteins, or mussel byssus silk proteins orvariants thereof, preferably the silk proteins are spider silk proteins.It is also contemplated that mixtures of two or more silk proteins areused. Alternatively, it is possible to add other polymers and/or fibrecomponents to the aqueous solution of step (a), (b), (c) and/or (d).Examples of such polymers include polyamide, polycaprolactone,polyacrylat, polyaramide, polylactic acid (PLA), polypropylene,polylactat, polyhydroxybutyrate, polyurethane, xanthan, cellulose,collagen, tropoelastin, elastin, keratin, cotton, wool or mixturesthereof as well as fibres made thereof.

In the embodiment, wherein another polymer is added to the silkprotein(s), it is preferred that the other polymer is also soluble inthe aqueous solution of step (a), (b) (c) or (d). It is more preferredthat at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% byweight of the fibre forming material in the silk protein spinning dopesolution is (are) silk proteins.

In the embodiment, wherein another polymer is added to the silkprotein(s), it is preferred that the silk protein spinning dope solutioncomprises at least 5% by weight, at least 10% by weight, at least 15% byweight, at least 20% by weight, at least 30% by weight, at least 40% byweight, or at least 50% by weight, and/or less than 50% by weight, lessthan 40% by weight, less than 30% by weight, less than 20% by weight, orless than 10% by weight of the other polymer. It is, thus, particularlypreferred that the content of the other polymer in the silk proteinspinning dope solution is in the range of between 5% and 50% by weight,between 5% and 30% by weight, or between 5% and 20% by weight.

The spider silk protein is preferably a major ampullate silk polypeptidesuch as a dragline silk polypeptide, a minor ampullate silk polypeptide,or a flagelliform silk polypeptide, preferably of an orb-web spider.

Preferred orb-web spiders comprise Araneus diadematus, Nephila spp. inparticular Nephila clavipes, Nephila senegalensis and Nephila edulis,and Lactrodectus hesperus. Preferred insects comprise Lepidoptera,particularly Bombycidae such as Bombyx mori or Hymenoptera, particularlyApoidea such as Anthophila.

It is preferred to use variants of such naturally occurring silkproteins, which have been optimized for their expression in heterologoushosts and for their fibre forming properties, e.g. by reducing the sizeand optimizing the amino acid composition. Such variants are preferablycharacterized by comprising naturally occurring repetitive units. It isalso preferred that such silk proteins or variants thereof areself-assembling. Self-assembling proteins have the ability to formordered macroscopic structures, e.g. fibrils or fibres. In contrast,protein aggregation generally forms amorphous unordered structures. Theability to self-assemble can be assessed, for example, by measuring oflight scattering and X-Ray diffraction. The skilled person is well awarehow to differentiate between a protein aggregate and the orderedstructure of an assembled protein. Thus, the skilled person trying toidentify a variant of a natural protein capable of self-assembly wouldbe required to introduce one or more alterations into the protein, e.g.deletions, mutations or additions within the boundaries set out in moredetail below, and investigate whether the formed structure possessesoriented and ordered properties caused by a self-assembling process asdetermined by light scattering and X-Ray. These measurements arepreferably conducted on fibres drawn from the silk protein dopesolution, which may be produced as set out in more detail below.

Preferably, the silk protein or variant thereof has a molecular weightof at least 20 kD and comprises at least two repetitive units eachcomprising at least one consensus sequence selected from the groupconsisting of:

-   (a) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably in    each case independently selected from the A, S, G, Y, P, and Q;-   (b) GGX, wherein X is any amino acid, preferably in each case    independently selected from Y, P, R, S, A, T, N and Q; and-   (c) A_(x), wherein x is an integer from 5 to 10.

The term a “repetitive unit”, as used herein, refers to a region whichcorresponds in amino acid sequence to a region that comprises orconsists of at least one peptide motif (e.g. AAAAAA (SEQ ID NO: 13) orGPGQQ (SEQ ID NO: 4)) that repetitively occurs within a naturallyoccurring silk polypeptide (e.g. MaSpI, ADF-3, ADF-4, or Flag) (i.e.identical amino acid sequence) or to an amino acid sequencesubstantially similar thereto (i.e. variation of amino acid sequence).In this regard “substantially similar” means a degree of amino acididentity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even99.9%, preferably over the whole length of the respective referencenaturally occurring amino acid sequence.

A “repetitive unit” having an amino acid sequence which is“substantially similar” to a corresponding amino acid sequence within anaturally occurring silk polypeptide (i.e. wild-type repetitive unit) isalso similar with respect to its functional properties, e.g. a silkpolypeptide comprising the “substantially similar repetitive unit” stillhas the ability to form a fibre. The skilled person can readily assesswhether the silk polypeptide comprising a “substantially similarrepetitive unit” is still capable of forming a fibre if he follows thedescription of how to produce a silk protein spinning dope solution andhow to form a fibre using such spinning dope as set out in theexperimental section.

A “repetitive unit” having an amino acid sequence which is “identical”to the amino acid sequence of a naturally occurring silk polypeptidecan, for example, be a portion of a silk polypeptide corresponding toone or more peptide motifs of MaSp I (SEQ ID NO: 43) MaSp II (SEQ ID NO:44), ADF-3 (SEQ ID NO: 1) and/or ADF-4 (SEQ ID NO: 2). A “repetitiveunit” having an amino acid sequence which is “substantially similar” tothe amino acid sequence of a naturally occurring silk polypeptide can,for example, be a portion of a silk polypeptide corresponding to one ormore peptide motifs of MaSpI (SEQ ID NO: 43) MaSpII (SEQ ID NO: 44),ADF-3 (SEQ ID NO: 1) and/or ADF-4 (SEQ ID NO: 2), but having one or moreamino acid substitution(s) at (a) specific amino acid position(s).

The term, a “repetitive unit”, as used herein, does not include thenon-repetitive hydrophilic amino acid domain generally thought to bepresent at the amino terminus and/or carboxyl terminus of naturallyoccurring silk polypeptides.

The term a “repetitive unit”, as used herein, preferably refers to anamino acid sequence with a length of 3 to 200 amino acids, or 5 to 150amino acids, preferably with a length of 10 to 100 amino acids, or 15 to80 amino acids and more preferably with a length of 18 to 60, or 20 to40 amino acids. For example, the repetitive unit according to thepresent invention can have a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, or 200 amino acids. More preferably, the repetitiveunit according to the invention consists of 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 18, 20, 24, 27, 28, 30, 34, 35, or 39 amino acids. In particularlypreferred embodiments, the silk protein comprises or consists of atleast 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably at least 95%and most preferably 100% of multiple copies of one identical repetitiveunit (e.g. A₂, Q6, or C₁₆, wherein the numerical 2, 6, or 16 representthe number of repetitive units) or multiple copies of two or moredifferent repetitive units (e.g. (AQ)₂₄, or (AQ)₁₂C₁₆). Said silkpolypeptide can further be modified by adding an artificial tag tofacilitate the detection or purification of said protein (e.g. T7 tag orHis Tag).

The repetitive unit of the silk polypeptide can comprise or consist ofan amino acid sequence of any region that comprises or consists of atleast one peptide motif that repetitively occurs within a naturallyoccurring silk polypeptide known to one skilled in the art. Preferably,the repetitive unit of the silk polypeptide comprises or consists of anamino acid sequence of a region that comprises or consists of at leastone peptide motif that repetitively occurs within an arthropod silkpolypeptide, more preferably within a spider silk polypeptide, or aninsect silk polypeptide. The repetitive unit of the silk polypeptide canalso comprise or consist of an amino acid sequence of a region thatcomprises or consists of at least one peptide motif that repetitivelyoccurs within a mussel silk polypeptide.

It is preferred that the spider silk repetitive unit comprises orconsists of an amino acid sequence of a region that comprises orconsists of at least one peptide motif that repetitively occurs within anaturally occurring major ampullate silk polypeptide (MaSp), such as adragline silk polypeptide, a minor ampullate silk polypeptide (MiSp), ora flagelliform (FLAG) silk polypeptide. Most preferably, the repetitiveunit comprises or consists of an amino acid sequence of a region thatcomprises or consists of at least one peptide motif that repetitivelyoccurs within a naturally occurring dragline silk polypeptide orflagelliform silk polypeptide.

It is also preferred that the insect silk repetitive unit comprises orconsists of an amino acid sequence of a region that comprises orconsists of at least one peptide motif that repetitively occurs within anaturally occurring silk polypeptide of Lepidoptera. More preferably,the insect silk repetitive unit comprises or consists of an amino acidsequence of a region that comprises or consists of at least one peptidemotif that repetitively occurs within a naturally occurring insect silkpolypeptide of Bombycidae, most preferably of Bombyx mori.

The term “consensus sequence”, as used herein, refers to an amino acidsequence which contains amino acids which frequently occur in a certainposition (e.g. “G”) and wherein, other amino acids which are not furtherdetermined are replaced by the place holder “X”.

Preferably, the silk protein comprises 2 to 100 repetitive units, i.e.at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60 or more repetitive units. The repetitive units inthe silk protein may be the same or different. It is preferred that thesame repetitive unit is used in one silk protein at least 2 times,preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, or 24 times. It has been observed that an increase ofthe length of the repetitive units increase the toughness of theresulting fibres. Accordingly, the molecular weight of the silk proteinmonomer is preferably between 10 kDa to 600 kDa, i.e. at least 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100kDa or smaller than 600, 590, 580, 570, 560, 550, 540, 530, 520, 510,500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370,360, 350, 340, 330, 320, 310, 300, 290, 280 270, 260, 250, 240, 230,220, 210, 200, 190, 180, 170, 160, or 150. It is, thus, particularlypreferred that the molecular weight is in the range of 40 kDa to 300kDa, more preferably 40 kDa to 200 kDa, more preferably 60 kDa to 200kDa, more preferably 80 kDa to 180 kDa, and even more preferably of 100kDa to 150 kDa.

In cases where the silk protein consists only of repetitive units themolecular weight of the silk protein will be as outlined above. In caseswherein the silk protein comprises further amino acid sequences, e.g.non-repetitive units and/or sequences intervening the repetitive units,e.g. linkers, the molecular weight of the silk protein will be larger.Similarly as for the molecular weight of the repetitive units within thesilk protein, it has been found that the overall molecular weight of thesilk protein improves the toughness of the resulting fibre. Accordingly,it is preferred that the monomer of the silk protein, comprising furtheramino acid sequences, preferably one or more non-repetitive units and/orsequences intervening the repetitive units, has a molecular weightbetween 20 kDa to 600 kDa, i.e. at least 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 kDa or smaller than 600, 590,580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450,440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310,300, 290, 280 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170,160, or 150. It is, thus, particularly preferred that the molecularweight is in the range of 40 kDa to 300 kDa, more preferably 40 kDa to200 kDa, more preferably 60 kDa to 200 kDa, more preferably 80 kDa to180 kDa, and even more preferably of 100 kDa to 150 kDa.

The iterated (peptide) motifs GPGXX (SEQ ID NO: 3) and GGX, i.e. glycinerich motifs, provide flexibility to the silk polypeptide and thus, tothe thread formed from the silk protein containing said motifs. Indetail, the iterated GPGXX (SEQ ID NO: 3) motif forms β-turn spiralstructures, which imparts elasticity to the silk polypeptide. Both majorampullate and flagelliform silks comprise a GPGXX (SEQ ID NO: 3) motif.The iterated GGX motif is associated with a helical structure havingthree amino acids per turn and is found in most spider silks. The GGXmotif may provide additional elastic properties to the silk. Theiterated polyalanine A_(x) (peptide) motif forms a crystalline β-sheetstructure that provides strength to the silk polypeptide. (WO03/057727). The GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19)(peptide) motifs have been selected from Resilin (WO 08/155304). Resilinis an elastomeric protein found in most arthropods (arthropoda). It islocated in specialised regions of the cuticle, providing low stiffnessand high strength (Elvin et al., Nature (473): 999-1002, 2005).

Preferred repetitive units comprise one A_(1x), wherein x is an integerfrom 5 to 10, i.e. 5, 6, 7, 8, 9, or 10, preferably 8, and one GPGXX(SEQ ID NO: 3), wherein X is any amino acid, preferably in each case isindependently selected from the A, S, G, Y, P, and Q, more preferably isQ in each instance. Another preferred repetitive unit comprises orconsists of at least 2, 3, or 4, preferably at least 4 consensussequences GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferablyin each case independently selected from A, S, G, Y, P, and Q.Preferably, X is in each instance Q.

Thus, in a preferred embodiment of the present invention, the silkpolypeptide comprises or consists of repetitive units each comprising atleast one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, or 9), preferably one, aminoacid sequence selected from the group consisting of GPGAS (SEQ ID NO:5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO: 8),GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGGG (SEQ ID NO: 10), GPGQG(SEQ ID NO: 40), and GPGGS (SEQ ID NO: 11). In a further preferredembodiment of the present invention, the silk polypeptide comprises orconsists of repetitive units each comprising at least one (e.g. 1, 2, 3,4, 5, 8, 7, or 8), preferably one, amino acid sequence selected from thegroup consisting of GGY, GGP, GGA, GGR, GGS, GGT, GGN, and GGQ. In anadditionally preferred embodiment of the present invention, the silkpolypeptide comprises or consists of repetitive units each comprising atleast one (e.g. 1, 2, 3, 4, 5, or 6), preferably one, amino acidsequence selected from the group consisting of AAAAA (SEQ ID NO: 12),AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ ID NO:15), AAAAAAAAA (SEQ ID NO: 16), and AAAAAAAAAA (SEQ ID NO: 17).

In another preferred embodiment of the invention, the silk polypeptidecomprises or consists of repetitive units each comprising at least one(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25), preferably one, amino acid sequence selectedfrom the group consisting of GPGAS (SEQ ID NO: 5), GPGSG (SEQ ID NO: 6),GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO: 8), GPGGA (SEQ ID NO: 9), GPGQQ(SEQ ID NO: 4), GPGGG (SEQ ID NO: 10), GPGQG (SEQ ID NO: 40), GPGGS (SEQID NO: 11), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO:12), AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ IDNO: 15), AAAAAAAAA (SEQ ID NO: 16), AAAAAAAAAA (SEQ ID NO: 17),GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19).

Most preferably, the silk polypeptide comprises, essentially consistsof, or consists of repetitive units, which comprise or consist of

-   -   i) GPGAS (SEQ ID NO: 5), AAAAAA (SEQ ID NO: 13), GGY, and GPGSG        (SEQ ID NO: 6) as amino acid sequence, preferably in this order,    -   ii) AAAAAAAA (SEQ ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ        ID NO: 7), and GPGGP (SEQ ID NO: 8) as amino acid sequence,        preferably in this order,    -   iii) GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID        NO: 4) and GPGQQ (SEQ ID NO: 4) as amino acid sequence,    -   iv) GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9), GGP, GPGGA        (SEQ ID NO: 9), and GGP as amino acid sequence, preferably in        this order,    -   v) AAAAAAAA (SEQ ID NO: 15), GPGQG (SEQ ID NO: 40), and GGR as        amino acid sequence, preferably in this order,    -   vi) AAAAAAAA (SEQ ID NO: 15), GPGGG (SEQ ID NO: 10), GGR, GGN,        and GGR as amino acid sequence, preferably in this order,    -   vii) GGA, GGA, GGA, GGS, GGA, and GGS as amino acid sequence,        preferably in this order, and/or    -   viii) GPGGA (SEQ ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID        NO: 11), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), and GPGGY        (SEQ ID NO: 7) as amino acid sequence, preferably in this order.

Thus, in a preferred embodiment, the repetitive units of the silkpolypeptide consist of module A: GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO:20), module C: GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 21),module Q: GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 22), module S:PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 25), module R:SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 26), or variantsthereof.

The silk protein may comprise combined repeats of only one of thesemodules or of combinations thereof. Preferred combinations arecharacterized as follows (the repetitive units are arranged from N- toC-terminus): XY, wherein X and Y are independently selected from A, C,Q, R and S or variant thereof and are each different, i.e. X and Y arenot C at the same time. Preferred combinations that are combined witheach other are CA, AC, CQ, QC, CS, SC, CR, RC, SR, RS, AQ, QA, AS, SA,AR, RA, QS, SQ, QR, RQ, SR, and RS. In further preferred combinationsblocks of three repetitive units are formed, which follow the followingconstruction scheme: XYZ, wherein X and Y are independently selectedfrom A, C, Q, R and S or variant thereof and are each different and Z isindependently selected from A, C, Q, R and S or variant thereof, ispreferably identical to X. Preferred combinations that are combined witheach other are CAA, CAC, CAQ, CAR, CAS, ACA, ACC. ACQ, ACR, ACS, CQA,CQC, CQQ, CQR, CQS, QCA, QCC, QCQ, QCR, QCS, CSA; CSC, CSQ, CSR, CSS,SCA, SCC, SCQ, SCR, SCS, CRA, CRC, CRQ, CRR, CRS, RCA, RCC, RCQ, RCR,RCS, SRA, SRC, SRQ, SRR, SRS, RSA, RSC, RSQ, RSR, RSS, AQA, AQC, AQQ,AQR, AQS, QAA, QAC, QAQ, QAR, QAS, ASA; ASC, ASQ, ASR, ASS, SAA, SAC,SAQ, SAR, SAS, ARA, ARC, ARQ, ARR; ARS, RAA, RAC, RAQ, RAR, RAS, QSA,QSC, QSQ, QSR, QSS, SQA, SQC, SQQ, SQR, SQS, QRA, QRC, QRQ, QRR, QRS,RQA, RQC, RQQ, RQR, RQS, SRA, SRC, SRQ, SRE, SRS, RSA, RSC, RSQ, RSR,and RSS. It is noted that it is in each case possible that one of therepetitive units is a variant of the respectively indicated repetitiveunit. Accordingly, preferred repetitive units comprised in the silkproteins used in the method of the invention follow the generalstructure X_(m), XY_(n) or XYZ_(o), wherein m is between 4 and 100, i.e.4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80 or more; n is between 2 and 60, i.e. 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60; and o isbetween 2 and 40, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, and 40.

The terms “combined with each other” or “concatenated with each other”,as used herein, mean that the modules (repetitive units) are directlycombined or concatenated with each other, or mean that the modules(repetitive units) are combined or concatenated with each other via oneor more spacer amino acids. Thus, in one embodiment, the modules(repetitive units) comprised in the silk polypeptide are directlycombined or concatenated with each other. In another embodiment, themodules (repetitive units) comprised in the silk polypeptide arecombined or concatenated with each other via one or more spacer aminoacids, preferably via 1 to 25 or 1 to 20 spacer amino acids, morepreferably via 1 to 15 or 1 to 10 spacer amino acids, and mostpreferably, via 1 to 5 spacer amino acids, e.g. via 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25spacer amino acids. Said spacer amino acid may be any amino acidnaturally occurring in proteins. Preferably, said spacer amino acid isnot proline. It is preferred that the spacer amino acid contains acharged group(s). Preferably, the spacer amino acid containing a chargedgroup(s) is independently selected from the group consisting ofaspartate, glutamate, histidine, and lysine. Said spacer amino acidshould be an amino acid which does not negatively affect the ability ofa silk polypeptide to form a fibre. Further, said spacer amino acidshould be an amino acid which does not cause steric hindrance, e.g. anamino acid having a small size such as lysine and cysteine. In morepreferred embodiments, the silk polypeptide comprises modules which aredirectly combined with each other and modules which are combined witheach other via 1 to 25 or 1 to 20 spacer amino acids, more preferablyvia 1 to 15 or 1 to 10 spacer amino acids, and most preferably, via 1 to5 spacer amino acids, e.g. via 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 spacer amino acids.

A module A, C, Q, S, or R variant differs from the reference (wild-type)module A, C, Q, S, or R from which it is derived by up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid changes in the aminoacid sequence (i.e. substitutions, additions, insertions, deletions,N-terminal truncations and/or C-terminal truncations). Such a modulevariant can alternatively or additionally be characterised by a certaindegree of sequence identity to the reference (wild-type) module fromwhich it is derived. Thus, a module A, C, Q, S, or R variant has asequence identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 99.9% to the respective reference (wild-type) module A, C, Q, S, orR set out above. Preferably, the sequence identity is over a continuousstretch of at least 10, 15, 18, 20, 24, 27, 28, 30, 34, 35, or moreamino acids, preferably over the whole length of the respectivereference (wild-type) module A, C, Q, S, or R.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference (wild-type) module A, C, Q, S, or R.It is further particularly preferred that the sequence identity is atleast 80% over a continuous stretch of at least 10, 15, 18, 20, 24, 28,or 30 amino acids, is at least 85% over a continuous stretch of at least10, 15, 18, 20, 24, 28, or 30 amino acids, is at least 90% over acontinuous stretch of at least 10, 15, 18, 20, 24, 28, or 30 aminoacids, is at least 95% over a continuous stretch of at least 10, 15, 18,20, 24, 28, or 30 amino acids, is at least 98% over a continuous stretchof at least 10, 15, 18, 20, 24, 28, or 30 amino acids, or is at least99% over a continuous stretch of at least 10, 15, 18, 20, 24, 28, or 30amino acids of the respective reference (wild-type) module A, C, Q, S,or R.

A fragment (or deletion variant) of module A, C, Q, S, or R haspreferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15 amino acids at its N-terminus and/or at its C-terminus.The deletion can also be internally.

Additionally, the module A, C, Q, S, or R variant or fragment is onlyregarded as a module A, C, Q, S, or R variant or fragment within thecontext of the present invention, if the modifications with respect tothe amino acid sequence on which the variant or fragment is based do notnegatively affect the ability of the silk polypeptide to self-assemble.The skilled person can readily assess whether the silk polypeptideself-assembles, for example, by measurement of light scattering and/orX-Ray diffraction.

It is more preferred that the repetitive units are independentlyselected from module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34). The modulesA^(C) (SEQ ID NO: 29), A^(K) (SEQ ID NO: 30), C^(C) (SEQ ID NO: 31),C^(K1) (SEQ ID NO: 32), C^(K2) (SEQ ID NO: 33) and C^(KC) (SEQ ID NO:34) are variants of the module A which is based on the amino acidsequence of ADF-3 of the spider Araneus diadematus and of module C whichis based on the amino acid sequence of ADF-4 of the spider Araneusdiadematus (WO 2007/025719). In module A^(C) (SEQ ID NO: 29) the aminoacid S (serine) at position 21 has been replaced by the amino acid C(cysteine), in module A^(K) (SEQ ID NO: 30) the amino acid S at position21 has been replaced by the amino acid K (lysine), in module C^(C) (SEQID NO: 31) the amino acid S at position 25 has been replaced by theamino acid 25 by C, in module C^(K1) (SEQ ID NO: 32) the amino acid S atposition 25 has been replaced by the amino acid K, in module C^(K2) (SEQID NO: 33) the amino acid E (glutamate) at position 20 has been replacedby the amino acid K, and in module C^(KC) (SEQ ID NO: 34) the amino acidE at position 20 has been replaced by the amino acid K and the aminoacid S at position 25 has been replaced by the amino acid C (WO2007/025719). Thus, in a more preferred embodiment, the repetitive unitsin the silk polypeptide consist of module A^(C):GPYGPGASAAAAAAGGYGPGCGQQ (SEQ ID NO: 29), module A^(K):GPYGPGASAAAAAAGGYGPGKGQQ (SEQ ID NO: 30), module C^(C):GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 31), module C^(K1):GSSAAAAAAAASGPGGYGPENQGPKGPGGYGPGGP (SEQ ID NO: 32), module C^(K2):GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 33), or module C^(KC):GSSAAAAAAAASGPGGYGPKNQGPCGPGGYGPGGP (SEQ ID NO: 34).

It has been observed that the toughness of the resulting fibre can beimproved, if non-repetitive units are included in the silk protein.Thus, at the same molecular weight a fibre produced from a silk proteinsolution comprising silk protein(s) comprising a non-repetitive unit islikely to have a higher toughness than fibres produced from a silkprotein solution comprising silk proteins without a non-repetitive unit.However, if the molecular weight of the silk proteins without anon-repetitive unit is increased a similar toughness of the fibre isachieved. Thus, it is preferred that fibres comprising silk proteinswithout one or more non-repetitive units have a higher molecular weight.The molecular weight is preferably increased by increasing the number ofrepetitive units in the silk molecule. It is preferred that a silkprotein without a non-repetitive unit have at least two, preferably atleast three, more preferably at least four, more preferably at leastfive and even more preferably at least six additional repetitive unitsin comparison to the protein comprising at least one non-repetitiveunit.

Most naturally occurring spider silk proteins also comprise at least onenon-repetitive unit. Therefore, the silk protein used in the method ofthe present invention preferably comprises at least one non-repetitive(NR) unit. The non-repetitive unit is preferably located N-terminally,C-terminally or N-terminally and C-terminally in the silk protein. Inthe context of the present invention, the term “non-repetitive (NR)unit” refers to a region of amino acids present in a naturally occurringsilk polypeptide that displays no obvious repetition pattern(non-repetitive unit or NR unit). NR units are protein domains with adefined tertiary structure in solution. Non-repetitive units preferablycomprise charged amino acids, e.g. Glu, Asp, Lys, or Arg, which allowthe formation of salt bridges between two proteins comprising anon-repetitive unit. Moreover, non-repetitive units often comprise oneor more Cys residues, which allow the formation of covalentintermolecular Cys-Cys bridges between two proteins comprising anon-repetitive unit. Without wishing to be bound by any theory theinventors believe that silk protein dimers formed by Cys-Cys bridgesfavour the assembling of the silk proteins into fibres. Preferably,non-repetitive units comprise at least 60, at least 70, at least 80, atleast 90 and more preferably at least 100 amino acids. Particularlypreferred ranges are between 100 and 200 amino acids. Preferably, theserepetitive units comprise at least one Cys residue.

The amino acid sequence of the non-repetitive unit corresponds to anon-repetitive amino acid sequence of naturally occurring draglinepolypeptides, preferably of ADF-3 (SEQ ID NO: 1) or ADF-4 (SEQ ID NO:2), or to an amino acid sequence substantially similar thereto. Theamino acid sequence of the non-repetitive unit may also correspond to anon-repetitive amino acid sequence of black widow. More preferably, theamino acid sequence of the non-repetitive unit corresponds to anon-repetitive carboxyl-terminal amino acid sequence of naturallyoccurring dragline polypeptides, preferably of ADF-3 (SEQ ID NO: 1) orADF-4 (SEQ ID NO: 2), or to an amino acid sequence substantially similarthereto. Even more preferably, the amino acid sequence of thenon-repetitive unit corresponds to a non-repetitive carboxyl-terminalamino acid sequence of a silk protein, preferably a spider silk proteinand even more preferably of ADF-3 (SEQ ID NO: 1) which comprises aminoacids 513 through 636, or of ADF-4 (SEQ ID NO: 2) which comprises aminoacids 302 through 410, or to an amino acid sequence substantiallysimilar thereto.

On the basis of above teaching and by sequence comparison the skilledperson is capable of identifying further non-repetitive units in silk anin particular in spider silk proteins that are suitable to be used inthe context of the method of the present invention.

In this regard “substantially similar” means a degree of amino acididentity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even99.9%, preferably over 20, 30, 40, 50, 60, 70, 80 or more amino acids,more preferably over the whole length of the respective referencenon-repetitive (carboxyl terminal) amino acid sequence of naturallyoccurring dragline polypeptides, preferably of ADF-3 (SEQ ID NO: 1) orADF-4 (SEQ ID NO: 2). A “non-repetitive unit” having an amino acidsequence which is “substantially similar” to a correspondingnon-repetitive (carboxyl terminal) amino acid sequence within anaturally occurring dragline polypeptide (i.e. wild-type non-repetitive(carboxyl terminal) unit), preferably within ADF-3 (SEQ ID NO: 1) orADF-4 (SEQ ID NO: 2), is also similar with respect to its functionalproperties, e.g. a silk polypeptide comprising the “substantiallysimilar non-repetitive unit” still has the ability to self-assemble. Theskilled person can readily assess whether the silk polypeptidecomprising the “substantially similar non-repetitive unit”self-assembles, for example, by measurement of light scattering and/orX-Ray diffraction.

Most preferably, the non-repetitive (NR) unit is NR3 (SEQ ID NO: 41);NR4 (SEQ ID NO: 42); NR5 (SEQ ID NO: 45); or NR6 (SEQ ID NO: 46); orvariants thereof. A NR3, NR4, NR5, or NR6 non-repetitive unit variantdiffers from the reference NR3 (SEQ ID NO: 41), NR4 (SEQ ID NO: 42), NR5(SEQ ID NO: 45); or NR6 (SEQ ID NO: 46) non-repetitive unit from whichit is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, or 30 amino acid changes in the amino acidsequence (i.e. exchanges, insertions, deletions, N-terminal truncationsand/or C-terminal truncations). Such a NR3, NR4, NR5, or NR6 unitvariant can alternatively or additionally be characterised by a certaindegree of sequence identity to the reference NR3, NR4, NR5, or NR6non-repetitive unit from which it is derived. Thus, a NR3, NR4, NR5, orNR6 non-repetitive unit variant has a sequence identity of at least 50%,55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 99.9% to the respective reference NR3, NR4, NR5, or NR6non-repetitive unit. Preferably, the sequence identity is over acontinuous stretch of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, ormore amino acids, preferably over the whole length of the respectivereference NR3, NR4, NR5, or NR6 non-repetitive unit.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference NR3, NR4, NR5, or NR6 non-repetitiveunit. It is further particularly preferred that the sequence identity isat least 80% over a continuous stretch of at least 20, 30, 40, 50, 60,70, or 80 amino acids, is at least 85% over a continuous stretch of atleast 20, 30, 40, 50, 60, 70, or 80 amino acids, is at least 90% over acontinuous stretch of at least 20, 30, 40, 50, 60, 70, or 80 aminoacids, is at least 95% over a continuous stretch of at least 20, 30, 40,50, 60, 70, or 80 amino acids, is at least 98% over a continuous stretchof at least 20, 30, 40, 50, 60, 70, or 80 amino acids, or is at least99% over a continuous stretch of at least 20, 30, 40, 50, 60, 70, or 80amino acids of the respective reference NR3, NR4, NR5, or NR6non-repetitive unit.

A fragment (or deletion variant) of a NR3, NR4, NR5, or NR6non-repetitive unit has preferably a deletion of up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55, or 60 amino acids at its N-terminus and/or atits C-terminus. The deletion can also be internally.

Additionally, the NR3, NR4, NR5, or NR6 non-repetitive unit variant orfragment is only regarded as a NR3, NR4, NR5, or NR6 non-repetitive unitvariant or fragment within the context of the present invention, if themodifications with respect to the amino acid sequence on which thevariant or fragment is based do not negatively affect the ability of asilk polypeptide to self-assemble. The skilled person can readily assesswhether the silk polypeptide comprising a NR3, NR4, NR5, or NR6non-repetitive unit variant or fragment self-assembles, for example, bymeasurement of light scattering and/or X-Ray diffraction.

It is particularly preferred that the silk protein used in the method ofthe invention is selected from the group consisting of ADF-3 (SEQ ID NO:1), ADF-4 (SEQ ID NO: 2), MaSp I (SEQ ID NO: 43), or MaSp II (SEQ ID NO:44); or variants thereof; or (C)_(m), (C)_(m)NR_(z), NR_(z)(C)_(m),NR_(z)(C)_(m)NR_(z), (AQ)_(n), (AQ)_(n)NR_(z), NR_(z)(AQ)_(n),NR_(z)(AQ)_(n)NR_(z), (QAQ)_(o), NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z),wherein m is an integer of 4 to 64, i.e. 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64; n isan integer of 10 to 40, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, or 40, o is an integer of 8 to 40, i.e. 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40; and z is an integer of 1 to 3, i.e.1, 2 or 3; and NR is in each case independently a non-repetitive unit,preferably NR3, NR4, NR5, or NR6 non-repetitive unit or variant thereof.

The above mentioned formulas are defined by one of the following: In theformula

-   i) (C)_(m), a “m” number of C modules, namely 8 to 64 C modules,    represented by the amino acid sequence according to SEQ ID NO: 21,    are combined with each other-   ii) (C)_(m)NR_(z), a “m” number of C modules, namely 8 to 48 C    modules, represented by the amino acid sequence according to SEQ ID    NO: 21, are combined with each other, wherein said C modules are    further combined with a “z” number of non-repetitive (NR) units,    namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive    (NR) units NR3 represented by the amino acid sequence according to    SEQ ID NO: 41, NR4 represented by the amino acid sequence according    to SEQ ID NO: 42, NR5 represented by the amino acid sequence    according to SEQ ID NO: 45, or NR6 represented by the amino acid    sequence according to SEQ ID NO: 46,    -   iii) NR_(z)(C)_(m), a “z” number of non-repetitive (NR) units,        namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive        (NR) units NR3 represented by the amino acid sequence according        to SEQ ID NO: 41, NR4 represented by the amino acid sequence        according to SEQ ID NO: 42, NR5 represented by the amino acid        sequence according to SEQ ID NO: 45, or NR6 represented by the        amino acid sequence according to SEQ ID NO: 46, is present (z=1)        or are combined with each other (z =2 or 3), wherein said        non-repetitive (NR) unit(s) is (are) further combined with a “m”        number of C modules, namely 2 to 64 C modules, represented by        the amino acid sequence according to SEQ ID NO: 21,-   iv) (AQ)_(n), a “n” number of A and Q module combinations, namely 6    to 36 A and Q module combinations, wherein module A is represented    by the amino acid sequence according to SEQ ID NO: 20 and module Q    is represented by the amino acid sequence according to SEQ ID NO:    22, are combined with each other,    -   v) (AQ)_(n)NR_(z), a “n” number of A and Q module combinations,        namely 10 to 40 A and Q module combinations, wherein module A is        represented by the amino acid sequence according to SEQ ID NO:        20 and module Q is represented by the amino acid sequence        according to SEQ ID NO: 22, are combined with each other, and        wherein said A and Q module combinations are further combined        with a “z” number of non-repetitive (NR) units, namely 1 to 3        non-repetitive (NR) units, e.g. the non-repetitive (NR) units        NR3 represented by the amino acid sequence according to SEQ ID        NO: 41, NR4 represented by the amino acid sequence according to        SEQ ID NO: 42, NR5 represented by the amino acid sequence        according to SEQ ID NO: 45, or NR6 represented by the amino acid        sequence according to SEQ ID NO: 46,-   vi) NR_(z)(AQ)_(n), a “z” number of non-repetitive (NR) units,    namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive    (NR) units NR3 represented by the amino acid sequence according to    SEQ ID NO: 41, NR4 represented by the amino acid sequence according    to SEQ ID NO: 42, NR5 represented by the amino acid sequence    according to SEQ ID NO: 45, or NR6 represented by the amino acid    sequence according to SEQ ID NO: 46, is present (z=1) or are    combined with each other (z=2 or 3), wherein said non-repetitive    (NR) unit(s) is (are) further combined with a “n” number of A and Q    module combinations, namely 10 to 40 A and Q module combinations,    wherein module A is represented by the amino acid sequence according    to SEQ ID NO: 20 and module Q is represented by the amino acid    sequence according to SEQ ID NO: 22,-   vii) (QAQ)_(o), a “o” number of Q, A and Q module combinations,    namely 8 to 24 Q, A and Q module combinations, wherein module Q is    represented by an amino acid sequence according to SEQ ID NO: 22 and    module A is represented by the amino acid sequence according to SEQ    ID NO: 20, are combined with each other,-   viii) (QAQ)_(o)NR_(z), a “o” number of Q, A and Q module    combinations, namely 8 to 16 Q, A and Q module combinations, wherein    module Q is represented by an amino acid sequence according to SEQ    ID NO: 22 and module A is represented by the amino acid sequence    according to SEQ ID NO: 20, are combined with each other, and    wherein said Q, A and Q module combinations are further combined    with a “z” number of non-repetitive (NR) units, namely 1 to 3    non-repetitive (NR) units, e.g. the non-repetitive (NR) units NR3    represented by the amino acid sequence according to SEQ ID NO: 41,    NR4 represented by the amino acid sequence according to SEQ ID NO:    42, NR5 represented by the amino acid sequence according to SEQ ID    NO: 45, or NR6 represented by the amino acid sequence according to    SEQ ID NO: 46, and-   ix) NR_(z)(QAQ)_(o), a “z” number of non-repetitive (NR) units,    namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive    (NR) units NR3 represented by the amino acid sequence according to    SEQ ID NO: 41, NR4 represented by the amino acid sequence according    to SEQ ID NO: 42, NR5 represented by the amino acid sequence    according to SEQ ID NO: 45, or NR6 represented by the amino acid    sequence according to SEQ ID NO: 46, is present (z=1) or are    combined with each other (z=2 or 3), wherein said non-repetitive    (NR) unit(s) is (are) further combined with a “o” number of Q, A and    Q module combinations, namely 8 to 40 Q, A and Q module    combinations, wherein module Q is represented by an amino acid    sequence according to SEQ ID NO: 22 and module A is represented by    the amino acid sequence according to SEQ ID NO: 20.

In the most preferred embodiments the silk protein that is used in themethod of the present invention is C₈NR4, C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3,(AQ)₂₄NR3, (AQ)₂₄, C₃₂, NR4C₁₆NR4, NR4C₃₂NR4, NR3C₁₆NR3, NR3C₃₂NR3,NR4(AQ)₁₂NR4, NR4(AQ)₂₄NR4, NR3(AQ)₁₂NR3, NR3 (AQ)₂₄NR3, (QAQ)₁₆,NR5C₁₆NR4, NR6C₁₆NR4, NR5C₃₂NR4, NR6C₃₂NR4, NR5C₁₆NR3, NR6C₁₆NR3,NR5C₂NR3, NR6C₁₆NR3, NR5(AQ)₁₂NR4, NR6(AQ)₁₂NR4, NR5(AQ)₂₄NR4,NR6(AQ)₂₄NR4, NR5(AQ)₁₂NR3, NR6(AQ)₁₂NR3, NR5(AQ)₂₄NR3, or NR6(AQ)₂₄NR3.

The denaturing agent serves the purpose of substantially unfolding thesilk proteins, i.e. to destroy the quaternary, tertiary and preferablyalso secondary structure of the silk protein. This allows inter alia thesolubilisation of insoluble recombinantly expressed silk protein and thesubsequent controlled transition of the denatured proteins into a state,which is suitable to form fibres of high toughness. The phrase that thedenaturing agent is comprised in the solution in a “silk proteindenaturing concentration” has to be understood to refer to aconcentration of the denaturing agent, in which the silk protein hassubstantially lost or lost its tertiary and preferably also itssecondary structure. Preferably, the protein is present in a so calledrandom coil structure. The skilled person is well aware of variousmethods of how to measure whether a given silk protein is denatured inthe solution in above outlined sense. These methods include inter aliaprotein nuclear magnetic resonance spectroscopy (protein NMR) andcircular dichroism (CD). The “silk protein denaturing concentration” fora given denaturing agent will also depend on the pH, the temperature andthe presence of other salts, e.g. buffers. The respectively requiredconcentration of a denaturing agent can be determined without undueburden for a given solution. The concentration of a given denaturantrequired to denature the silk protein to the extent required in thecontext of the method of the invention will also depend on the furtherconditions in the aqueous solution of step (a). Preferably, thetemperature of the aqueous solution is between 4° C. and 30° C., morepreferably between 15° C. and 25° C. and/or the pH is between pH 5 and9, preferably between 6 and 8, It is also preferred that salts arepresent in a concentration of between 0.01 to 1 M, preferably of between0.1 and 0.5 M. Thus, the concentration of the respective denaturingagent is preferably selected to denature the silk protein under aboveindicated preferred conditions.

It has been observed by the present inventors that guanidinium salts areparticularly suitable as denaturing agents in the context of step (a) ofthe method of the present invention. The most preferred denaturant fordenaturing the silk protein in step (a) is guanidinium thiocyanate.

For guanidinium salts preferred protein denaturing concentrations are inthe range of 5 M to 8 M. These concentrations are preferably employedwhen the aqueous solution provided in step (a) has a temperature ofbetween 4° C. and 30° C., more preferably between 15° C. and 25° C.and/or the pH is between pH 5 and 9, preferably between pH 6 and pH 8and/or salts are present in a concentration of between 0.01 to 1 M.

It is preferred that the aqueous solution provided in step (a) comprisesa buffer to adapt the pH. Suitable buffers include Tris-HCl, Hepes,MOPS, phosphate-buffer, NaHCO₃/Na₂CO₃. The buffers are provided inconcentrations typical for protein solutions. Preferred salts that maybe comprised in the aqueous solution provided in step (a) comprise NaCl,and/or KCl Preferred concentrations of such salts are between 0.1 to 0.5M.

The concentration of the silk protein in the aqueous solution providedin step (a) is chosen such that it is below the desired concentration inthe silk protein spinning dope solution, which is the result of step(d). Preferably, it is in the range of 4 to 15% w/v, i.e. 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15, more preferably in the range of 6 to 12%w/v.

The present inventors have determined that it is advantageous forobtaining the state of the silk protein that allows the formation offibres with high toughness, if the guanidinium salt concentration, whichmay be comprised in aqueous solution provided in step (a) is reducedprior to step (d), e.g. in an additional dialysis step after step (c)but prior to step (d) or during step (d) at least 100-fold, preferablyat least 200-fold.

The purpose of step (d) is the increase of the concentration of thespider silk to a concentration that is suitable for producing fibres.Such concentration is preferably at least 10% w/v, more preferably atleast 12% w/v, more preferably at least 15% w/v, more preferably atleast 20% w/v, more preferably at least 25% w/v. The removal of thedenaturing agent in steps (b) and (c) may be continued in step (d).

It is preferred that a chemical chaperone is present in the aqueous silkprotein solution in step (a), (b), and/or (c) or the spinning dopesolution produced in step (d). The term “chemical chaperone” refers inthe context of the present invention to molecules which aid proteinfolding and/or increase protein solubility. The chemical chaperonesodium phenylbutyrate, for example, appears to act by masking ofhydrophobic domains that have formed in the proteins during folding.Through these effects chemical chaperones counteract the tendency ofproteins in solution to aggregate (see e.g. Bathaie, B. B. et al. (2011)The Protein J. 30 (7), 480-489). Preferred chemical chaperones that maybe used in the context of the method of the present invention comprisedimethylsulfoxide (DMSO), polyamine, like e.g. spermine or spermidine,polyole, like glycerine, urea, mono or disaccharides, e.g. trehalose,cholic acid, sodium phenylbutyrate or trimethylamine N-oxide. For thepurpose of this invention, urea in a concentration up to 1 M is definedas a chemical chaperone. Urea is a particularly preferred chemicalchaperone, which acts as a chemical chaperon at concentration of lessthan 1 M. Preferably, the aqueous silk protein solutions in step (a),(b) and/or (c) or the silk protein spinning dope solution of step (d)comprise the chemical chaperone at a concentration of less than 1 M,preferably between 0.25 and 0.75 M. The presence of a chemical chaperoneappears to stabilize the state of the silk protein that is capable offorming fibres of high toughness, thus, it is particularly preferredthat the chemical chaperone is present in step (d) and in the resultingsilk protein spinning dope solution.

It is preferred that steps (b), (c) and (d) are carried out subsequentlyin this order. However, depending on the method used to reduce theconcentration of the protein denaturant in steps (b) and (c) the silkprotein concentration may be increased at the same time. While it ispreferred that the concentration is not significantly increased in steps(b) and (c), in one embodiment the concentration is increased at thesame time when reducing the denaturant concentration. Thus, in oneembodiment steps (b) and (d) and/or steps (c) and (d) are carried outconcomitantly.

The reducing of the concentration of the protein denaturant in steps (b)and/or (c) can be carried out by any art known method. Preferably it iscarried out by dialysis and/or diafiltration.

Dialysis is typically carried out using a dialysis membrane with adefined molecular weight cut of that retains the silk protein on oneside of the dialysis membrane and separates it from the dialysissolution on the other side of the membrane. Typically an excess ofdialysis solution is provided. It is preferred that this solutioncomprises the components, e.g. denaturants, at the concentration desiredas endpoint of the respective dialysis step, e.g. if the denaturantconcentration is reduced 8-fold to 14-fold the concentration of thedenaturant in the dialysis solution is 8-fold to 14-fold lower than theconcentration in the aqueous solution provided in step (a). Accordingly,dialysis allows changing the composition of the aqueous solutioncomprising the silk protein by providing a dialysis buffer of thedesired composition, e.g. the pH may be altered or the saltconcentration may be increased, while reducing the concentration of thedenaturant. Typically, there is also a small change in the volume of thedialysed aqueous silk protein solution, which may lead to an increase inthe concentration of the silk protein in the aqueous solution. Thischange may be compensated for by the addition of aqueous solution. It ispreferred that aqueous solution is added with a composition similar oridentical to the dialysis solution. It is also possible to add aqueoussolution, which is similar or identical to the dialysis solution but forthe concentration of the denaturant. The later approach may allow a morerapid decrease of the concentration of the denaturant, preferably of theguanidinium salt.

In another preferred embodiment the concentration of the denaturant isreduced by diafiltration, preferably by tangential flow filtration(TFF). In TFF, typically, the silk protein comprising solution flowsparallel to the filter membrane. A pressure differential across themembrane causes fluid and filterable solutes (whose molecular weight issmaller than that of the membranes or behaves in this way, such asglobular proteins) to flow through the filter. Diafiltration can beeither discontinuous or continuous diafiltration, e.g. TFF. Indiscontinuous diafiltration, the solution is concentrated, and the lostvolume is replaced by a new aqueous solution. Preferably, the lostvolume is continuously replaced by new aqueous solution to minimize orprevent concentration of the silk proteins in the aqueous solution. Incontinuous diafiltration, the solution volume is maintained by theinflow of new buffer solution while the old buffer solution is removed.In both cases it is preferred that the aqueous solution comprises thedenaturant to be removed, preferably the guanidinium salt in the desiredend concentration of the respective step, i.e. step (b), step (c) and/orstep (d).

The reduction of the concentration of the denaturant in step (b) ispreferably by 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold or14-fold. Thus, if the concentration of the denaturant in the aqueoussolution provided in step (a) is 7 M than it is preferred that theconcentration at the completion of step (b) is between 0.875 M and 0.5M.

During removal of the denaturant the silk protein partially enters anequilibrium state reflecting the respective denaturant concentration inthe aqueous solution. This a process that requires some time and it is,therefore, preferred that step (b) is carried out for at least 30 min,more preferably for at least 1 h, more preferably for at least 2 h, morepreferably for at least 4 h, more preferably for at least 6 h, morepreferably for at least 8 h, more preferably for at least 10 h. Fordialysis the equilibrium state may be achieved by allowing the aqueoussilk protein sample to contact the dialysis solution for the indicatedperiod of time. For diafiltration the equilibrium state may be achievedby choosing the conditions of diafiltration in such that the respectivereduction of the denaturant concentration is gradually achieved over theindicated time periods or preferred time periods.

In a preferred embodiment the concentration of the denaturant is between0.6 M to 0.4 M at the end of step (b). In particular if the denaturantused in step (a) is a guanidinium salt the guanidinium saltconcentration is reduced to between 0.6 M to 0.4 M, preferably to 0.5 Mat the end of step (b).

It is preferred that step (b) is carried out at a temperature between 4°C. and 30° C., preferably between 15° C. and 25° C.

It is preferred that the concentration of the components of the aqueoussolution with the exception of the denaturant are not altered duringstep (b), although the silk protein concentration may vary slightlydepending on the composition of the dialysis buffer or the conditions ofthe diafiltration.

The reduction of the concentration of the denaturant in step (c) ispreferably by 1.5-fold, 1.6-fold, 1.7,-fold, 1.8-fold, 1.9-fold,2.0-fold, 2.1-fold, 2.2.-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold,2.7-fold, 2.8-fold, 2.9-fold or 3-fold. To provide the silk protein withsufficient time to enter the equilibrium state it is preferred that step(c) is carried out for at least 30 min, more preferably for at least 1h, more preferably for at least 2 h, more preferably for at least 4 h,more preferably for at least 6 h, more preferably for at least 8 h, morepreferably for at least 10 h.

It is preferred that step (c) is carried out at a temperature between 4°C. and 30° C., preferably between 15° C. and 25° C.

In particular if the denaturant used in step (a) is a guanidinium saltthe guanidinium salt concentration is reduced to between 0.3 M to 0.2 M,preferably to about 0.25 M at the end of step (c).

The concentrating in step (d) can be achieved by any method known in theart for increasing the concentration of a protein in aqueous solution.In a particularly useful embodiment, the silk protein is concentrated bydialysis or by filtration. Dialysing is preferably carried out against adehydrating solution such as a solution comprising a hygroscopicpolymer. Examples of suitable hygroscopic polymers include, but are notlimited to, polyethylene glycol (PEG), amylase, and sericin, or acombination of two or more thereof. PEG molecules are available in arange of molecular sizes and the selection of the PEG will be determinedby the membrane chosen for dialysis and the rate of concentrationrequired. Preferably, the PEG is of a molecular weight of about 8,000 toabout 10,000 g/mol and has a concentration of about 25% to about 50%. Insome embodiments, the separation can be conducted bymembrane-filtration, which includes, but is not limited to, methods suchas single pass, dead-end, direct flow filtration (DFF), and cross-flowor tangential flow filtration (TFF). Filtration is based on theprinciple of separating molecules according to size using asemi-permeable membrane of a defined range of pore sizes. It is known tothose skilled in the art that combinations of filtration methods andmembrane types may be used in separation. According to the invention,membrane-filtration is the separation of cellular components effected bypolymeric or inorganic membranes. Within the art, there are fourcommonly accepted categories of membranes defined by the size of thematerial they remove from the carrier liquid. Methods of sequentiallyfiltering through membranes from the smallest to largest pore size areReverse Osmosis (RO), Nanofiltration (NF), Ultrafiltration (UF), andMicrofiltration (MF). Filtration with the above-mentioned membranesseparates molecules according to their molecular weight by usingmembranes with specific pore sizes. For example, separation with ROmembranes that have pore sizes less than 0.001 micrometers is intendedto separate molecules that have a molecular weight less than 200Daltons. Filtration with NF membranes that have pore sizes from0.001-0.008 micrometers, inclusive, is intended to separate moleculesthat have a molecular weight from 200 D to 15 kDa inclusive. Filtrationwith UF membranes that have 30 pore sizes from 0.005-0.1 micrometers,inclusive, is intended to separate molecules that have a molecularweight from 5 kDa-300 kDa, inclusive. Filtration with microfiltrationmembranes that have pore sizes from 0.05-3.0 micrometers, inclusive, isintended to separate molecules that have a molecular weight from 100 kDa3000 kDa and larger. Membrane-filtration can separate the solubilisedsilk proteins from other components based on size exclusion by utilizingmembranes that have a particular Molecular Weight Cut-Off (MQWCO) thatis determined by the pore size of the membrane. The MWCO, also calledNominal Molecular Weight Limit (NMWL) or Nominal Molecular WeightCut-Off (NMWCO), is the kilo Dalton size designation for the filtrationby membranes. The MWCO is defined as the molecular weight of themolecule that is 90% retained by the membrane. Because, for example,molecules of the same molecular weight can have significantly differentshapes, the MWCO is not an exact metric, but is nevertheless a usefulmetric and is commonly employed by filter manufacturers. Bothhydrophobic as well as hydrophilic membranes may be used. Such membranesmay be used as flat sheets or in a spirally wound configuration. Hollowfibres may also be used. In relation to compositions of UF membranes,any number of potential membrane materials may be used including, butnot limited to, regenerated cellulose, polyether sulfone (which may ormay not be modified to alter its inherent hydrophobicity),polyvinylidene fluoride, and ceramic and metal oxide aggregates. Manypolyether sulfone UF membranes can withstand a pH range of 0.5-13, andtemperatures ranging 15 up to 85° C. Materials for MF membranes includeeverything used for UF membranes, as well as polycarbonate,polypropylene, polyethylene and PTFE (TEFLON™). In a preferredembodiment, TFF is used to both concentrate the silk proteins and toalter the buffer composition. Thus, in a preferred embodiment theguanidinium salt concentration is reduced as outlined above and thechemical chaperone, preferably urea, concentration is increased toobtain the above outlined preferred chemical chaperone concentration ofthe silk protein spinning dope produced in step (d).

In another preferred embodiment phase separation is used to effectconcentration of the silk protein solution in step (d). This is based onthe phenomenon observed by the present inventors that aqueous silkprotein solutions have the tendency to separate into a silk proteinenriched aqueous silk protein phase and a silk protein depleted aqueoussilk protein phase. The former phase will localize at the bottom of avessel containing the aqueous silk protein solution resulting from step(c) or from the optional removal step which further removes thedenaturant present in a silk protein denaturing concentration in step(a), e.g. the guanidinium salt. To allow phase separation to occur it ispreferred that the silk protein solution resulting from the denaturantremoval steps is maintained for at least 2 h, preferably for at least 4h, more preferably for at least 8 h. To facilitate the phase separationminimal disturbance of the solution is preferred.

The aqueous solution of at least one of the steps (a), (b), (c) or (d)may further contain a basic amino acid, i.e. lysine, arginine, orglutamine, preferably arginine. Basic amino acids both have pH bufferingcapacity and tend to stabilize proteins in solution. It is preferredthat the basic amino acid, preferably arginine is comprised at aconcentration between 1 mM and 1 M. Preferred concentrations are in therange of 10 mM to 250 mM.

In a preferred embodiment of the first aspect of the invention themethod further comprises the step of producing a fibre by drawing thefibre from the silk protein spinning dope solution, by extruding thesilk protein spinning dope solution, or by a combination of these twotechnologies.

“Extrusion” means a process of pushing a solution through adie/opening/nozzle by applying pressure before the die/opening/nozzle.“Drawing” means a process of passing the solution through adie/opening/nozzle by applying pressure after the die/opening/nozzle,whereby the pressure after the die/opening exceed the pressure beforethe die/opening/nozzle. This can be obtained by drawing gravity,negative pressure or the use of a venturi-nozzle.

The silk protein spinning dope can be spun together with other polymers.Examples include, but are not limited to, polymers (e.g., polypropylene,polyamide, polyester), fibres and silks of other plant and animalsources. A preferred embodiment is silk protein fibre blended with 10%by weight of polyamide. In a further preferred embodiment, the silkprotein fibre is blended with polyamide, polyaramide, polylactic acid(PLA), polypropylene, polycaprolactone, polyacrylat, polylactat,polyhydroxybutyrate, polyurethane, xanthan, cellulose, natural andrecombinant collagen, keratin, natural and recombinant tropoelastin,elastin, cotton, wool or mixtures thereof. Preferably, the content ofthis polymer (e.g., polypropylene, polyamide, polyester) in theresulting fibre is less than 50% by weight, more preferably less then40% by weight, less than 30% by weight, less than 20% by weight and evenmore preferably less than 15% by weight. Alternatively, it is preferredthat the content of this polymer (e.g., polypropylene, polyamide,polyester) in the resulting fibre is at least 5% by weight, at least 10%by weight, at least 15% by weight, at least 20% by weight, at least 30%by weight, at least 40% by weight, or at least 50% by weight, and/orless than 50% by weight, less than 40% by weight, less than 30% byweight, less than 20% by weight, or less than 10% by weight. It is,thus, particularly preferred that the content of this polymer (e.g.,polypropylene, polyamide, polyester) in the resulting fibre is in therange of between 5% and 50% by weight, between 5% and 30% by weight, orbetween 5% and 20% by weight. The production of such combinations offibres can be readily practiced to enhance any desired characteristics,e.g., appearance, softness, weight, durability, water-repellentproperties, improved cost-of-manufacture, that may be generally soughtin the manufacture and production of fibres for medical, industrial, orcommercial applications. The silk protein fibres can further be bundled,braided or woven with other fibre types.

In a preferred embodiment of the extrusion technology the silk proteinspinning dope solution is extruded directly into a coagulation bath,e.g. the spinneret or the die/opening/nozzle may be submerged in acoagulation bath. Similarly, it is possible to immerse the drawn fibrein a coagulation bath after the fibre has formed, e.g. immediatelybehind the drawing nozzle (the spinneret or the die/opening/nozzle islocated above a coagulation bath). The coagulation bath preferablycomprises phosphate buffer, and/or alcohols. Preferred alcohols arelinear or branched C₁ to C₆ mono or di-alcohols, preferably ethanol orisopropanol. The concentration of the alcohol(s) in the coagulation bathis preferably in the range of 50 to 100% w/v, e.g. 75% or 90% w/v.

The inventors have discovered that the toughness of the fibre can besignificantly improved, if the fibre is extended after drawing orextrusion. Without wishing to be bound by any theory it is believed thatthe extension leads to an alignment and more regular distribution of thesilk protein molecules within the fibre and thereby improves theproperties of the fibre. It is preferred that the fibre is extendedafter it has been drawn or extruded. Such extension can be carried outin a continuous or discontinuous process. In the continuous process itis preferred that the fibre is exposed to a pulling force. Preferably,the extension of the fibre is by at least 2-fold in comparison to thelength of the fibre as drawn or extruded, preferably the extension is atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, and more preferably at least 10-fold. Preferably, suchextension is carried out in the presence of the coagulation solution,e.g. the fibre is at least partially submerged in the coagulationsolution. This solution is also referred to as stretching solution inthe context of the present invention. It has the same composition as thecoagulation solution.

The skilled person is well aware of various methods to apply a definedpulling force to achieve the above outlined extension. If, for example,the fibre is drawn or extruded from the nozzle of the spinneret with aspeed of 10 cm/s and the fibre is subsequently wound up with a speed of1 m/s, the extension will be at least 10-fold. The skilled person iswell aware of various methods to stretch an extruded fibre in apredetermined way. For example, a roller may be positioned behind thenozzle that draws out the fibre with a speed of 1 m/s the next rollermoves with a speed of 2 m/s and subsequent rollers may have an evenhigher speed, which will lead to an incremental increase of thestretching. The fibre may also be intermittently relaxed to produce aseries of stretching and relaxing motions. As outlined above thefoldness of extension is calculated on the basis of the fibre as drawnor extruded and the product at the end of the stretching (and possiblerelaxing) process.

During extension the cross-sectional area of the fibre is reduced. It ispreferred that extension leads to a reduction of the cross-sectionalarea of at least 10%, preferably of at least 20%, of at least 30%, of atleast 40%, of at least 50%, 60%, of at least 65%, and more preferably ofat least 70%.

The thickness (diameter) of the fibre upon extrusion is preferably inthe range of 5 μm to 200 μm and more preferably in the range of 20 μm to150 μm. Alternatively, the thickness (diameter) of the fibre uponextrusion is preferably in the range of 30 μm to 90 μm and morepreferably in the range of 40 μm to 80 μm, or the thickness (diameter)of the fibre upon extrusion is preferably in the range of 110 μm to 200μm and more preferably in the range of 110 μm to 150 μm. After theextension, the thickness (diameter) of the fibre is preferably in therange of 1 μm to 100 μm and more preferably in the range of 1 μm to 50μm. Alternatively, the thickness (diameter) of the fibre is preferablyin the range of 30 μm to 90 μm and more preferably in the range of 40 μmto 80 μm after the extension. It is preferred that the fibre thickness(diameter) is uniform or has a variation rate of up to 5%, e.g. up to1%, 2%, 3%, 4%, or 5%. In the latter case, the reference to a fibrethickness (diameter) in the present invention refers to a fibre averagethickness (diameter).

In a second aspect the present invention provides a method for producinga fibre comprising the steps:

-   -   (i) providing the silk spinning dope solution producible by the        method of the first aspect of the invention; and    -   (ii) producing a fibre by drawing from or extruding or        combination thereof the silk protein spinning dope solution.

The provision of the silk spinning dope solution is preferably carriedout as described in the context of the first aspect of the invention. Inparticular it is preferred that the silk protein spinning dope solutionis extruded into a coagulation bath.

It is also preferred that the fibre is extended in a subsequentstretching step as outlined regarding the first aspect of the presentinvention.

In a third aspect the present invention provides a spinning dopesolution producible by the method of the first aspect of the invention.

In a fourth aspect the present invention relates to a fibre producibleby the method of the first or second aspect of the invention. This fibreexhibits a toughness (measured in MJ/m³) that is superior to thetoughness of prior art fibres comprising silk proteins of similar oridentical molecular weight. Without wishing to be bound by any theorythe inventors believe that this is due to the unique three dimensionalstructures that the silk proteins can attain if the fibres are drawnfrom a silk protein spinning dope as provided by the method of the firstaspect of the present invention. Preferably, at least 10% by weight ofthe material of the fibre is (are) silk protein(s). It is more preferredthat at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% byweight of the material of the fibre is (are) silk proteins.

If the fibre comprises 100% by weight of silk proteins, it is preferredthat these proteins are not naturally occurring silk proteins.

It is also preferred that the silk protein monomer(s) comprised in thefibre has (have) a molecular weight in the range of between 20 kDa to600 kDa, i.e. at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 kDa or smaller than 600, 590, 580, 570, 560, 550,540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410,400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280 270,260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, or 150. It is,thus, particularly preferred that the molecular weight is in the rangeof 40 kDa to 300 kDa, more preferably 40 kDa to 200 kDa, morepreferably, and even more preferably of 100 kDa to 150 kDa.

In cases where the silk protein dimerize, preferably via disulfide bondsin the NR region it is preferred that the another embodiment themolecular weight of the protein dimer is in the range of between 20 kDato 600 kDa, i.e. at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or200 kD or smaller than 600, 590, 580, 570, 560, 550, 540, 530, 520, 510,500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370,360, 350, 340, 330, 320, 310 or 300, most preferably between 200 kDa to300 kD. Preferably, the fibre has a toughness (MJ/m³) that is theproduct of the molecular weight of the silk protein(s) in kDa and afactor of at least 1.0. This relation is obtained at least up to amolecular weight of the silk protein(s) of 300 kDa and is at least 300MJ/m³ for proteins with a molecular weight of above 300 kDa. Preferably,the factor is at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.

If the silk protein solution comprises more than one silk protein themolecular weight of the silk proteins for the purpose of thiscalculation is determined by the molecular weight of the silk proteinwith the lowest molecular weight.

For the purpose of calculating the molecular weight of the silk proteinscomprised in the silk protein solution in cases wherein the silkproteins are capable of dimerizing through covalent bonds, e.g.disulfide bonds between two Cys residues, the weight of thenon-dimerized monomer is used. Thus, fibres of the invention, whichcomprise dimers with a molecular weight of 200 kD, the toughness iscalculated on the basis of the molecular weight of the monomers formingthe dimers, i.e. 100 kD. Thus, in the example the fibre has a toughnessof at least 100 MJ/m³.

The fibre is preferably extended. It is preferred that the extension isby at least 2-fold in comparison to the length of the fibre as drawn orextruded, preferably the extension is at least 4-fold, at least 5-fold,at least 6-fold, at least 7-fold, at least 8-fold, and more preferablyat least 10-fold.

During extension the cross-sectional area of the fibre is reduced. It ispreferred that extension leads to a reduction of the cross-sectionalarea of at least 10%, preferably of at least 20%, of at least 30%, of atleast 40%, of at least 50%, 60%, of at least 65%, and more preferably ofat least 70%.

The thickness (diameter) of the fibre upon extrusion is preferably inthe range of 5 μm to 200 μm and more preferably in the range of 20 μm to150 gm. Alternatively, the thickness (diameter) of the fibre uponextrusion is preferably in the range of 30 μm to 90 μm and morepreferably in the range of 40 μm to 80 μm, or the thickness (diameter)of the fibre upon extrusion is preferably in the range of 110 μm to 200μm and more preferably in the range of 110 μm to 150 μm. After theextension, the thickness (diameter) of the fibre is preferably in therange of 1 μm to 100 μm and more preferably in the range of 1 μm to 50μm. Alternatively, the thickness (diameter) of the fibre is preferablyin the range of 30 μm to 90 μm and more preferably in the range of 40 μmto 80 μm after the extension. It is preferred that the fibre thickness(diameter) is uniform or has a variation rate of up to 5%, e.g. up to1%, 2%, 3%, 4%, or 5%. In the latter case, the reference to a fibrethickness (diameter) in the present invention refers to a fibre averagethickness (diameter).

In a fifth aspect the present invention relates to a fibre comprising atleast one silk protein, wherein at least 10% by weight of the materialof the fibre is(are) silk protein(s), wherein the silk proteinmonomer(s) has have a molecular weight in the range of 20 kDa to 600 kDaand the fibre has a toughness (MJ/m³) that is the product of themolecular weight of the silk protein(s) in kDa and the factor of atleast 1.0 at least up to an molecular weight of the silk protein(s) of300 kDa and is at least 300 MJ/m³ for proteins with an molecular weightof above 300 kDa.

Preferably, the factor is at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.

It is more preferred that at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% or 100% by weight of the material of the fibre is (are) silkproteins.

If the fibre comprises 100% by weight of silk proteins, it is preferredthat these proteins are not naturally occurring silk proteins.

The silk proteins that may be comprised in the fibre according to thefifth aspect of the invention are those described as suitable in thecontext of the first aspect of the invention, including all thepreferred and particularly preferred embodiments. Accordingly, it ispreferred that the silk protein comprises 2 to 100 repetitive units,i.e. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60 or more repetitive units.

It is particularly preferred that the silk protein comprises at leasttwo repetitive units each comprising at least one consensus sequenceselected from the group consisting of:

-   (a) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably in    each case independently selected from the A, S, G, Y, P, and Q;-   (b) GGX, wherein X is any amino acid, preferably in each case    independently selected from Y, P, R, S, A, T, N and Q; and-   (c) A_(x), wherein x is an integer from 5 to 10.

Preferably, the repetitive units are independently selected from moduleA (SEQ ID NO: 20), module C (SEQ ID NO: 21), module Q (SEQ ID NO: 22),module S (SEQ ID NO: 25), module R(SEQ ID NO: 26), or variants thereof.

For example, the silk protein comprises 2 to 100 repetitive units, i.e.at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60 or more repetitive units, wherein the repetitiveunits are independently selected from module A (SEQ ID NO: 20), module C(SEQ ID NO: 21), module Q (SEQ ID NO: 22), module S (SEQ ID NO: 25),module R(SEQ ID NO: 26), or variants thereof.

Preferably, the silk protein further comprises at least onenon-repetitive (NR) unit.

Preferred NR units are NR3 (SEQ ID NO: 41), NR4 (SEQ ID NO: 42), NR5(SEQ ID NO: 45) or NR6 (SEQ ID NO: 46) or variants thereof.

The silk protein may be selected from the group consisting of ADF-3 (SEQID NO: 1), ADF-4 (SEQ ID NO: 2), MaSp I (SEQ ID NO: 43), or MaSp II (SEQID NO: 44); or variants thereof; or (C)_(m)NR_(z), NR_(z)(C)_(m),NR_(z)(C)_(m)NR_(z), (AQ)_(n)NR_(z), NR_(z)(AQ)_(n),NR_(z)(AQ)_(n)NR_(z), (NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z), wherein m is aninteger of 10 to 64, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59 or 60; n is an integer of 10 to 40, i.e. 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40; o is an integer of 8 to 40, i.e. 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40; and z is aninteger of 1 to 3,i.e. 1, 2 or 3, preferably 1 and NR in each caseindependently is a non-repetitive unit.

The silk protein is preferably C₈NR4, C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3,(AQ)₂₄NR3, ((AQ)₂₄C32, NR4C₁₆NR4, NR4C₃₂NR4, NR3C₁₆NR3, NR3C₃₂NR3,NR4(AQ)₁₂NR4, NR4(AQ)₂₄NR4, NR3(AQ)₁₂NR3, NR3 (AQ)₂₄NR3, (QAQ)₁₆,NR5C₁₆NR4, NR6C₁₆NR4, NR5C₃₂NR4, NR6C₃₂NR4, NR5C₁₆NR3, NR6C₁₆NR3,NR5C₃₂NR3, NR6C₁₆NR3, NR5(AQ)₁₂NR4, NR6(AQ)₁₂NR4, NR5(AQ)₂₄NR4,NR6(AQ)₂₄NR4, NR5(AQ)₁₂NR3, NR6(AQ)₁₂NR3, NR5(AQ)₂₄NR3, NR6(AQ)₂₄NR3.

The fibre is preferably extended. It is preferred that the extension isby at least 2-fold in comparison to the length of the fibre as drawn orextruded, preferably the extension is at least 4-fold, at least 5-fold,at least 6-fold, at least 7-fold, at least 8-fold, and more preferablyat least 10-fold. The extension significantly improves the toughness ofthe fibre.

The thickness (diameter) of the fibre upon extrusion is preferably inthe range of 5 μm to 200 μm and more preferably in the range of 20 μm to150 μm. Alternatively, the thickness (diameter) of the fibre uponextrusion is preferably in the range of 30 μm to 90 μm and morepreferably in the range of 40 μm to 80 μm, or the thickness (diameter)of the fibre upon extrusion is preferably in the range of 110 μm to 200μm and more preferably in the range of 110 μm to 150 μm. After theextension, the thickness (diameter) of the fibre is preferably in therange of 1 μm to 100 μm and more preferably in the range of 1 μm to 50μm. Alternatively, the thickness (diameter) of the fibre is preferablyin the range of 30 μm to 90 μm and more preferably in the range of 40 μmto 80 μm after the extension. It is preferred that the fibre thickness(diameter) is uniform or has a variation rate of up to 5%, e.g. up to1%, 2%, 3%, 4%, or 5%. In the latter case, the reference to a fibrethickness (diameter) in the present invention refers to a fibre averagethickness (diameter).

The fibre of the fourth or fifth aspect of the invention may furthercomprise at least one additional polymer. For example, the fibre of thefourth or fifth aspect of the invention may comprise a synthetic and/ornatural polymer. Preferred polymers comprise artificial and/or naturallyoccurring polymers including polyamide, polycaprolactone, polyacrylate,polylactate, polyhydroxybutyrate, polyurethane, xanthan, cellulose,collagen, tropoelastin, elastin, keratin, cotton, wool or mixturesthereof. Preferably, the content of the at least one additional polymer,e.g. synthetic and/or natural polymer, in the fibre is less than 50% byweight, more preferably less then 40% by weight, less than 30% byweight, less than 20% by weight and even more preferably less than 15%by weight. Alternatively, it is preferred that the content of the atleast one additional polymer, e.g. synthetic and/or natural polymer, inthe fibre is at least 5% by weight, at least 10% by weight, at least 15%by weight, at least 20% by weight, at least 30% by weight, at least 40%by weight, or at least 50% by weight, and/or less than 50% by weight,less than 40% by weight, less than 30% by weight, less than 20% byweight, or less than 10% by weight. It is, thus, particularly preferredthat the content of the at least one additional polymer, e.g. syntheticand/or natural polymer, in the fibre is in the range of between 5% and50% by weight, between 5% and 30% by weight, or between 5% and 20% byweight. The production of such fibre can be readily practiced to enhanceany desired characteristics, e.g., appearance, softness, weight,durability, water-repellent properties, improved cost-of-manufacture,that may be generally sought in the manufacture and production of fibresfor medical, industrial, or commercial applications. The silk proteinfibres can further be bundled, braided or woven with other fibre types.

As mentioned above, it is more preferred that at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95% or 100% by weight of the material of thefibre is (are) silk protein(s). It is even more preferred that at least10% by weight of the material of the fibre is (are) silk protein(s) andno more than 90% by weight of the material of the fibre is anotherpolymer, e.g. synthetic or natural polymer, at least 20% by weight ofthe material of the fibre is (are) silk protein(s) and no more than 80%by weight of the material of the fibre is another polymer, e.g.synthetic or natural polymer, at least 30% by weight of the material ofthe fibre is (are) silk protein(s) and no more than 70% by weight of thematerial of the fibre is another polymer, e.g. synthetic or naturalpolymer, at least 40% by weight of the material of the fibre is (are)silk protein(s) and no more than 60% by weight of the material of thefibre is another polymer, e.g. synthetic or natural polymer, at least50% by weight of the material of the fibre is (are) silk protein(s) andno more than 50% by weight of the material of the fibre is anotherpolymer, e.g. synthetic or natural polymer, at least 60% by weight ofthe material of the fibre is (are) silk protein(s) and no more than 40%by weight of the material of the fibre is another polymer, e.g.synthetic or natural polymer, at least 70% by weight of the material ofthe fibre is (are) silk protein(s) and no more than 30% by weight of thematerial of the fibre is another polymer, e.g. synthetic or naturalpolymer, at least 80% by weight of the material of the fibre is (are)silk protein(s) and no more than 20% by weight of the material of thefibre is another material, e.g. synthetic or natural polymer, at least90% by weight of the material of the fibre is (are) silk protein(s) andno more than 10% by weight of the material of the fibre is anotherpolymer, e.g. synthetic or natural polymer, or at least 95% by weight ofthe material of the fibre is (are) silk protein(s) and no more than 5%by weight of the material of the fibre is another polymer, e.g.synthetic or natural polymer.

EXAMPLES Spinning Process:

-   1. Preparation of the spinning dope

500 mg of the recombinant spider silk protein (C₁₆NR4, C₃₂NR4,(AQ)₁₂NR3, NR5(AQ)₁₂NR3, or (AQ)₂₄NR3)) was dissolved in 10 mL of 6 MGdmSCN (5% (w/v)). After the protein was dissolved, insoluble parts wereremoved by centrifuging (8500 rpm, 30 min, 18° C.). The supernatant wasdialyzed (MWCO: 6-8 kDa) each time for 4 hours with the followingbuffers:

-   1) Buffer 1: 50 mM NH₄HCO₃ (pH 7.8), 500 mM urea, 500 mM GdmSCN-   2) Buffer 2: 50 mM NH₄HCO₃ (pH 7.8), 500 mM urea, 250 mM GdmSCN-   3) Buffer 3: 50 mM NH₄HCO₃ (pH 7.8), 500 mM urea    As a next step eADF4 C-proteins (C₁₆NR4, C32NR4) were handled    differently than eADF3 AQ-proteins ((AQ)₁₂NR3, NR5(AQ)₁₂NR3,    (AQ)₂₄NR3):-   C-proteins: dialysis against 20% (w/v) PEG (35 kDa), 500 mM urea    until a concentration of 15% is reached. The spinning dope can now    be used for spinning-   AQ-proteins: transfer to 15 mL Greiner tubes. The tubes were kept at    4° C. overnight, where phase separation takes place. The    concentrated (lower) phase (9-15% (w/v)) was used for spinning-   2. Wet-Spinning

The spinning dope was transferred into a 1 mL syringe with a 22Gcannula. The filled syringe was mounted on a syringe pump. The cannulawas bent down perpendicular to the syringe so that the tip of thecannula is submerged into the coagulation bath

Coagulation/Stretching Baths:

-   C-proteins: 90% Isopropanol, 10% H₂O-   AQ-proteins: 75% Isopropanol, 25% H₂O,    The spinning dope was extruded into the coagulation bath with a    spinning speed of 5 μL/min. The coagulated fiber was taken out of    the bath and was stretched in a stretching bath. Coagulation and    stretching bath are identical for the respective C- or AQ-protein.    After stretching, the fibers were taken out of the stretching bath    and placed in a clean petri dish to dry in open air for at least 2    h.-   3. Tensile Testing

For tensile tests, the dried fibers were cut into 1 cm fragments, whichwere glued onto plastic frames (gauge length: 2 mm) using plastic glue.The glued fiber-fragments were air-dried overnight. The diameter of thefiber-fragments was determined with a light microscope (100-fold,200-fold and 400-fold magnification) at three points distributed evenlythroughout the fiber-fragment.

Afterwards, stress-strain curves were recorded on a tensile tester witha 0.5 N load cell at a relative humidity of 30%. The fibers wereextended with a rate of 0.04 mm/s until they ruptured. The results areshown in Table 1

TABLE 1 Experimental results of different spider silk proteins. Thetable further shows a comparison to experimental data regarding fourrecombinant spider silk protein fibers described in Xia et al. (2010)Native-sized recombinant spider silk protein produced in metabolicallyengineered Escherichia coli results in a strong fiber. PNAS August 10,2010 Vol. 107 no. 3214059-14063. Young's Stretching DiameterExtensibility Strength Toughness modulus Protein [%] [μm] [%] [MPa][MJ/m³] [GPa] eADF4 C₁₆NR4 300 23 76 366 162.8 1.4 56 kDa monomer C₃₂NR4254.0 104 kDa monomer eADF3 (AQ)₁₂NR3 500 47 ± 4 124.6 ± 47.5 206.8 ±56.4  97.4 ± 37   2.5 ± 0.4 58 kDa monomer NR5 (AQ)₁₂NR3 600 22 ± 1 59.5 ± 22.5 350.0 ± 39.0  98.4 ± 41.0 4.2 ± 0.2 72 kDa monomer(AQ)₂₄NR3 600 14 ± 2 118.2 ± 29.2 205.9 ± 39.3 150.0 ± 49.9 2.2 ± 0.7106 kDa monomer Xia et 16-mer 2.5 70 1 al. (*) 54 kDa 32-mer 3 200 3 100kDa 64-mer 5 270 10 10 192 kDa 96-mer 20 600 141 23 284 kDa (*) The dataregarding toughness were calculated on the basis of FIG. 3 of Xia et. al(supra).

1. Method for producing a silk protein spinning dope solution comprisingthe steps: (a) providing an aqueous solution comprising at least onesilk protein and a protein denaturant or mixture of protein denaturantsat a silk protein denaturing concentration, wherein the totalconcentration of the silk protein(s) in the solution is less than 20%w/v; (b) reducing the concentration of the protein denaturant by 8-foldto 14-fold; (c) reducing the concentration of the protein denaturant by1.5 to 3-fold; and (d) producing the silk protein spinning dope solutionby concentrating the silk protein(s) in the solution at least 1.5-foldin comparison to its(their) concentration in step (a) to a concentrationof at least 10% w/v.
 2. The method of claim 1, wherein the silk proteinis a spider silk protein, preferably a dragline or flagelliform proteinof an orb-web spider (Araneidae); insect silk protein, or mussel byssussilk protein or a variant thereof.
 3. (canceled)
 4. The method of claim2, wherein the silk protein or variant thereof has a molecular weight ofat least 20 kDa and comprises at least two repetitive units eachcomprising at least one consensus sequence selected from the groupconsisting of: (a) GPGXX (SEQ ID NO: 3), wherein X is any amino acid,preferably in each case independently selected from the A, S, G, Y, P,and Q; (b) GGX, wherein X is any amino acid, preferably in each caseindependently selected from Y, P, R, S, A, T, N and Q; and (c) A_(x),wherein x is an integer from 5 to
 10. 5. The method of claim 4, whereinthe repetitive units are independently selected from module A (SEQ IDNO: 20), module C (SEQ ID NO: 21), module Q (SEQ ID NO: 22), module S(SEQ ID NO: 25), module R (SEQ ID NO: 26) or variants thereof.
 6. Themethod of claim 1, wherein the silk protein comprises at least onenon-repetitive (NR) unit.
 7. The method of claim 6, wherein the NR unitis NR3 (SEQ ID NO: 41), NR4 (SEQ ID NO: 42), NR5 (SEQ ID NO: 45) or NR6(SEQ ID NO: 46) or variants thereof.
 8. (canceled)
 9. (canceled)
 10. Themethod of claim 1, wherein the denaturing agent comprises a guanidiniumsalt.
 11. (canceled)
 12. The method of claim 10, wherein the proteindenaturing concentration is in the range of 5 M to 8 M for theguanidinium salt.
 13. (canceled)
 14. The method of claim 1, wherein theaqueous solution in step (a), (b), and/or (c) or the spinning dopesolution produced in step (d) comprises a chemical chaperone at aconcentration of less than 1 M, preferably between 0.25 and 0.75 M. 15.The method claim 14, wherein the chemical chaperone is dimethylsulfoxide(DMSO), polyamine, a polyol, urea, cholic acid, a mono or disaccharide,sodium phenylbutyrate or trimethylamine N-oxide or combinations thereof.16. (canceled)
 17. (canceled)
 18. The method of claim 1, wherein theaqueous solution of at least one of the steps (a)-(d) further containslysine, glutamine, or arginine, preferably at a concentration between 1mM and 1 M.
 19. The method of claim 1, further comprising the step ofproducing a fibre by drawing from, extruding, or a combination thereofthe silk protein spinning dope solution.
 20. (canceled)
 21. (canceled)22. Method for producing a fibre comprising the steps: (i) providing thesilk spinning dope solution producible by the method of claim 1; and(ii) producing a fibre by drawing or extruding or combination thereoffrom the silk protein spinning dope solution.
 23. (canceled) 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. Fibre comprising at leastone silk protein, wherein at least 10% by weight of the material of thefibre is (are) silk protein(s), wherein the silk protein monomer(s)has(have) a molecular weight in the range of 20 kDa to 600 kDa and thefibre has a toughness (MJ/m³) that is the product of the molecularweight of the silk protein(s) in kDa and the factor of at least 1.0 atleast up to a molecular weight of the silk protein(s) of 300 kDa and isat least 300 MJ/m³ for proteins with a molecular weight of above 300kDa.
 28. The fibre of claim 27, wherein the silk protein comprises atleast two repetitive units each comprising at least one consensussequence selected from the group consisting of: (a) GPGXX (SEQ ID NO:3), wherein X is any amino acid, preferably in each case independentlyselected from the A, S, G, Y, P, and Q; (b) GGX, wherein X is any aminoacid, preferably in each case independently selected from Y, P, R, S, A,T, N and Q; and (c) A_(x), wherein x is an integer from 5 to
 10. 29. Thefibre of claim 28, wherein the repetitive units are independentlyselected from module A (SEQ ID NO: 20), module C (SEQ ID NO: 21), moduleQ (SEQ ID NO: 22), module S (SEQ ID NO: 25), module R (SEQ ID NO: 26),or variants thereof.
 30. The fibre of claim 28, wherein the silk proteinfurther comprises at least one non-repetitive (NR) unit.
 31. The fibreof claim 30, wherein the NR unit is NR3 (SEQ ID NO: 41), NR4 (SEQ ID NO:42), NR5 (SEQ ID NO: 45) or NR6 (SEQ ID NO: 46) or variants thereof. 32.(canceled)
 33. (canceled)
 34. The fibre of claim 27, wherein the fibrefurther comprises a synthetic or natural polymer.
 35. The fibre of claim34, wherein the polymer is polyamide, polycaprolactone, polyacrylate,polyaramide, polylactic acid (PLA), polypropylene, polylacetate,polyhydroxybutyrate, polyurethane, xanthan, cellulose, collagen,tropoelastin, elastin, keratin, cotton, wool or mixtures thereof.